Ana Rita C. Duarte

In the tissue engineering (TE) field, the concept of producing multifunctional scaffolds, capable not only of acting as templates for cell transplantation but also of delivering bioactive agents in a controlled manner, is an emerging strategy aimed to enhance tissue regeneration. In this work, a complex hybrid release system consisting in a three-dimensional (3D) structure based on poly(d,l-lactic acid) (PDLLA) impregnated with chitosan/chondroitin sulfate nanoparticles (NPs) was developed. The scaffolds were prepared by supercritical fluid foaming at 200 bar and 35 °C, and were then characterized by scanning electron microscopy (SEM) and micro-CT. SEM also allowed to assess the distribution of the NPs within the structure, showing that the particles could be found in different areas of the scaffold, indicating a homogeneous distribution within the 3D structure. Water uptake and weight loss measurements were also carried out and the results obtained demonstrated that weight loss was not significantly enhanced although the entrapment of the NPs in the 3D structure clearly enhances the swelling of the structure. Moreover, the hybrid porous biomaterial displayed adequate mechanical properties for cell adhesion and support. The possibility of using this scaffold as a multifunctional material was further evaluated by the incorporation of a model protein, bovine serum albumin (BSA), either directly into the PDLLA foam or in the NPs that were eventually included in the scaffold. The obtained results show that it is possible to achieve different release kinetics, suggesting that this system is a promising candidate for dual protein delivery system for TE applications. © 2010 Elsevier B.V.

This work addresses the preparation of 3D porous scaffolds of blends of chitosan and poly(l-lactic acid), CHT and PLLA, using supercritical fluid technology. Supercritical assisted phase-inversion was used to prepare scaffolds for tissue engineering purposes. The physicochemical and biological properties of chitosan make it an excellent material for the preparation of drug delivery systems and for the development of new biomedical applications in many fields from skin to bone or cartilage regeneration. On the other hand, PLLA is a synthetic biodegradable polymer widely used for biomedical applications. Supercritical assisted phase-inversion experiments were carried out in samples with different polymer ratios and different polymer solution concentrations. The effect of CHT:PLLA ratio and polymer concentration and on the morphology and topography of the scaffolds was assessed by SEM and Micro-CT. Infra-red spectroscopic imaging analysis of the scaffolds allowed a better understanding on the distribution of the two polymers within the matrix. This work demonstrates that supercritical fluid technology constitutes a new processing technology, clean and environmentally friendly for the preparation of scaffolds for tissue engineering using these materials. © 2010 Elsevier B.V.

Electrospun structures were proposed as scaffolds owing to their morphological and structural similarities with the extracellular matrix found in many native tissues. These fibrous structures were also proposed as drug release systems by exploiting the direct dependence of the release rate of a drug on the surface area. An osteogenic differentiation factor, dexamethasone (DEX), was incorporated into electrospun polycaprolactone (PCL) nanofibers at different concentrations (5, 10, 15 and 20 wt.{%} polymer), in a single-step process. The DEX incorporated into the polymeric carrier is in amorphous state, as det ermined by DSC, and does not influence the typical nanofibers morphology. In vitro drug release studies demonstrated that the dexamethasone release was sustained over a period of 15 days. The bioactivity of the released dexamethasone was assessed by cultivating human bone marrow mesenchymal stem cells (hBMSCs) on 15 wt.{%} DEX-loaded PCL NFMs, under dexamethasone-absent osteogenic differentiation medium formulation. An increased concentration of alkaline phosphatase and deposition of a mineralized matrix was observed. Phenotypic and genotypic expression of osteoblastic-specific markers corroborates the osteogenic activity of the loaded growth/differentiation factor. Overall data suggests that the electrospun biodegradable nanofibers can be used as carriers for the sustained release of growth/differentiation factors relevant for bone tissue engineering strategies. © 2010 Elsevier Ltd.

The aim of this study was to evaluate the possibility of preparing chitosan porous matrixes using supercritical fluid technology. Supercritical immersion precipitation technique was used to prepare scaffolds of a natural biocompatible polymer, chitosan, for tissue engineering purposes. The physicochemical and biological properties of chitosan make it an excellent material for the preparation of drug delivery systems and for the development of new biomedical applications in many fields from skin to bone or cartilage. Immersion precipitation experiments were carried out at different operational conditions in order to optimize the processing method. The effect of different organic solvents on the morphology of the scaffolds was assessed. Additionally, different parameters that influence the process were tested and the effect of the processing variables such as polymer concentration, temperature and pressure in the chitosan scaffold morphology, porosity and interconnectivity was evaluated by micro computed tomography. The preparation of a highly porous and interconnected structure of a natural material, chitosan, using a clean and environmentally friendly technology constitutes a new processing technology for the preparation of scaffolds for tissue engineering using these materials. © (2010) Trans Tech Publications.

In this work, a starch-based polymer, namely a blend of starch-poly(epsilon-caprolactone) was processed by supercritical assisted phase inversion process. This processing technique has been proposed for the development of 3D structures with potential applications in tissue engineering applications, as scaffolds. The use of carbon dioxide as non-solvent in the phase inversion process leads to the formation of a porous and interconnected structure, dry and free of any residual solvent. Different processing conditions such as pressure (from 80 up to 150 bar) and temperature (45 and 55 degrees C) were studied and the effect on the morphological features of the scaffolds was evaluated by scanning electron microscopy and micro-computed tomography. The mechanical properties of the SPCL scaffolds prepared were also studied. Additionally, in this work, the in vitro biological performance of the scaffolds was studied. Cell adhesion and morphology, viability and proliferation was assessed and the results suggest that the materials prepared are allow cell attachment and promote cell proliferation having thus potential to be used in some for biomedical applications.

The aim of this study was to evaluate the possibility of preparing dexamethasone-loaded starch-based porous matrices in a one-step process. Supercritical phase inversion technique was used to prepare composite scaffolds of dexamethasone and a polymeric blend of starch and poly(l-lactic acid) (SPLA) for tissue engineering purposes. Dexamethasone is used in osteogenic media to direct the differentiation of stem cells towards the osteogenic lineage. Samples with different drug concentrations (5-15 wt.{%} polymer) were prepared at 200 bar and 55 °C. The presence of dexamethasone did not affect the porosity or interconnectivity of the polymeric matrices. Water uptake and degradation studies were also performed on SPLA scaffolds. We conclude that SPLA matrices prepared by supercritical phase inversion have a swelling degree of nearly 90{%} and the material presents a weight loss of ∼25{%} after 21 days in solution. Furthermore, in vitro drug release studies were carried out and the results show that a sustained release of dexamethasone was achieved over 21 days. The fitting of the power law to the experimental data demonstrated that drug release is governed by an anomalous transport, i.e., both the drug diffusion and the swelling of the matrix influence the release of dexamethasone out of the scaffold. The kinetic constant was also determined. This study reports the feasibility of using supercritical fluid technology to process in one step a porous matrix loaded with a pharmaceutical agent for tissue engineering purposes. © 2009 Acta Materialia Inc.

Supercritical fluid technology has proven to be useful for many pharmaceutical applications and is now emerging as an alternative to conventional processes for the preparation of 3D structures and injectable particles suitable to be used in regenerative medicine. A current overview of the basic principles underlying supercritical fluid technology, the state of the art and future potential of this technology are presented.

Supercritical fluid impregnation was tested to prepare a new scaffold loaded with a bioactive compound. Dexamethasone is used in osteogenic media to direct the differentiation of stem cells towards the osteogenic lineage. Dexamethasone was impregnated in chitosan scaffolds at different operating conditions, in order to optimize the impregnation process. Pressure and temperature affect the carbon dioxide density and influence the swelling of the polymer and the drug solubility in the fluid phase, therefore these are two important parameters that were studied in this work. Chitosan sponges prepared by freeze drying were impregnated with the active compound at pressures from 8.0 up to 14.0 MPa and temperatures from 35 up to 55 °C. The effect of the impregnation contact time (3 h and 6 h) was also evaluated. From the experiments performed we can conclude that the yield of impregnation is lower when increasing pressure and temperature. The contact time will mainly influence the amount of drug impregnated in the scaffold and for higher contact times the impregnation yield is also higher. Scanning electron microscopy shows particles of dexamethasone in the bulk of the scaffold, which confirms the feasibility of the supercritical fluid impregnation technology for the preparation of delivery devices. The loading capacity of the scaffolds was determined by spectroscopic analysis and the highest loading was achieved for the experiments performed at 8.0 MPa and 35 °C. Furthermore, in vitro drug release studies were carried out and the results show that dexamethasone was sustainably released. Supercritical fluid impregnation proved to be feasible for the preparation of a drug delivery system for bone tissue engineering purposes. © 2008 Elsevier Ltd. All rights reserved.

The aim of this study was to evaluate the possibility of preparing starch-based porous matrixes using supercritical fluid technology. Supercritical immersion precipitation technique was used to prepare scaffolds of a polymeric blend of starch and poly(l-lactic acid) for tissue engineering purposes.Immersion precipitation experiments were carried out at different operational conditions and highly porous and interconnected scaffolds were obtained. Two organic solvents, dichloromethane and chloroform were tested, and from the results obtained chloroform was the more favourable for the process. The effect of polymer solution concentration (5 up to 20 wt{%}), temperature (35 up to 55 °C) and pressure (100 up to 200 bar) in the SPLA (50:50 wt{%}) membrane morphology, porosity and interconnectivity was evaluated. All the conditions tested were in the region of total miscibility between the organic solvent and carbon dioxide. Additionally, a blend with a different starch-poly(l-lactic acid) ratio (30:70 wt{%}) was tested. Bicontinuous structures were formed indicating that the L-L demixing process that governs the phase inversion is the spinodal decomposition. © 2008 Elsevier B.V. All rights reserved.

The aim of this study was to develop a new process for the production of bioactive 3D scaffolds using a clean and environmentally friendly technology. The possibility of preparing composite scaffolds of Bioglass?? and a polymeric blend of starch and poly(l-lactic acid) (SPLA50) was evaluated. Supercritical phase-inversion technique was used to prepare inorganic particles loaded starch-based porous composite matrixes in a one-step process for bone tissue engineering purposes. Due to their osteoconductive properties some glasses and ceramics are interesting materials to be used for bone tissue engineering purposes; however their poor mechanical properties create the need of a polymeric support where the inorganic fraction can be dispersed. Samples impregnated with different concentrations of Bioglass?? (10 and 15{%} wt/wt polymer) were prepared at 200??bar and 55????C. The presence of Bioglass?? did not affect the porosity or interconnectivity of the polymeric matrixes. Dynamic mechanical analysis has proven that the modulus of the SPLA50 scaffolds increases when glass particles are impregnated within the matrix. In vitro bioactivity studies were carried out using simulated body fluid and the results show that a calcium-phosphate layer started to be formed after only 1??day of immersion. Chemical analysis of the apatite layer formed on the surface of the scaffold was performed by different techniques, namely EDS and FTIR spectroscopy and X-ray diffraction (XRD). The ion concentration in the simulated body fluid was also carried out by ICP analysis. Results suggest that a bone-like apatite layer was formed. This study reports the feasibility of using supercritical fluid technology to process, in one step, a porous matrix loaded with a bioactive material for tissue engineering purposes. ?? 2009 Elsevier B.V. All rights reserved.

Over the past several years, the definition of a scaffold for tissue engineering has changed dramatically, from a material that acts only as an inert structural support for cell attachment to serving as a more complex and dynamic environment for tissue development. This paper is a review on the existing and on the new emerging techniques based on supercritical fluid technology for the preparation of scaffolds and particles for tissue engineering applications. Supercritical fluid technology has already proven to be feasible for many pharmaceutical applications and is now emerging as an alternative to conventional materials' processing methods for the preparation of three-dimensional structures and injectable particles suitable to be used in regenerative medicine. The basic principles underlying each technique are here presented as well as the advantages and disadvantages of each process. The state of the art is reviewed and the major conclusions of the studies reported in the literature are discussed.

In this experimental phase equilibrium study, we show for the first time that it is possible to stabilize structure sH of hydrogen clathrate hydrate with the help of some selected promoters. It was established that the formation pressures of these systems are significantly higher than that of structure sII of hydrogen clathrate hydrate when tetrahydrofuran (THF) is used as a promoter. Although no experimental evidence is available yet, it is estimated that the hydrogen storage capacity of structure sH can be as high as 1.4 wt {%} of H 2 , which is about 40{%} higher compared to the hydrogen storage capacity in structure sH. © 2008 American Chemical Society.

The possibility of preparation of ophthalmic drug delivery systems using compressed anti-solvent technology was evaluated. Eudragit RS 100 and RL 100 were used as drug carriers, acetazolamide was the model drug processed. Compressed anti-solvent experiments were carried out as a semi-continuous or a batch operation from a liquid solution of polymer(s) + solute dissolved in acetone. Both techniques allowed the recovery of composite particles, but the semi-continuous operation yielded smaller and less aggregated populations than the batch operation. The release behaviour of acetazolamide from the prepared microparticles was studied and most products exhibited a slower release than the single drug. Moreover, the release could be controlled to some extent by varying the ratio of the two Eudragit used in the formulation and by selecting one or the other anti-solvent technique. Simple diffusion models satisfactorily described the release profiles. Composites specifically produced by semi-continuous technique have a drug release rate controlled by a diffusion mechanism, whereas for composites produced by the batch operation, the polymer swelling also contributes to the overall transport mechanism. © 2006 Elsevier B.V. All rights reserved.

The possibility of preparation of ophthalmic drug delivery systems using compressed anti-solvent technology was evaluated. Eudragit RS 100 and RL 100 were used as drug carriers, acetazolamide was the model drug processed. Compressed anti-solvent experiments were carried out as a semi-continuous or a batch operation from a liquid solution of polymer(s) + solute dissolved in acetone. Both techniques allowed the recovery of composite particles, but the semi-continuous operation yielded smaller and less aggregated populations than the batch operation. The release behaviour of acetazolamide from the prepared microparticles was studied and most products exhibited a slower release than the single drug. Moreover, the release could be controlled to some extent by varying the ratio of the two Eudragit used in the formulation and by selecting one or the other anti-solvent technique. Simple diffusion models satisfactorily described the release profiles. Composites specifically produced by semi-continuous technique have a drug release rate controlled by a diffusion mechanism, whereas for composites produced by the batch operation, the polymer swelling also contributes to the overall transport mechanism. © 2006 Elsevier B.V. All rights reserved.